GFP technology for the study of biocontrol agents in tritrophic interactions: a case study with Pseudozyma flocculosa

GFP technology was applied to the biocontrol agent (BCA) Pseudozyma flocculosa to study its development and interactions at the tritrophic level plant-powdery mildew-BCA. Transformation experiments with GFP led to the production of a strongly fluorescent strain, Act-4, that displayed biocontrol traits typical of P. flocculosa WT. Following inundative applications, growth of P. flocculosa Act-4 was closely and almost exclusively associated with the colonies of the pathogen regardless of the powdery mildew species or the host plant tested. Development of P. flocculosa Act-4 on control leaves alone was extremely limited 24 h after its application and was typical of the epiphytic growth characterizing this type of yeast-like fungus. Based on the strong correlation between the colonization pattern of the different powdery mildew species tested and the presence of P. flocculosa Act-4, as determined by its fluorescence, it seems that growth of the BCA is dependant on the presence of powdery mildews. These results demonstrate that the GFP technology can be used to study plant-pathogen-BCA interactions and fulfill a wide array of purposes ranging from fundamental observations of the biocontrol behavior of a BCA to very applied ones serving some of the requirements for the registration of BCA's such as defining their environmental fate.     

The effectivity of Tilletiopsis albescens in biocontrol of powdery mildew

Tilletiopsis albescens grows well on powdery mildew fungi inoculated on barley or cucumber leaves and causes collapse of the colonies. Application of ballistospores or cut mycelium was equally effective for biocontrol, and the effectiveness tended to increase exponentially with the concentration of germinating units (conidia and cut mycelium) applied. Seventy percent relative humidity or more is required for effective biocontrol. Two applications of T. albescens in the period from 3 days before to 3 days after inoculation with powdery mildew were more effective than one. Applications before inoculation or 7 days after inoculation with powdery mildew had little effect. T. albescens followed the powdery mildew as it was disseminated to uninoculated leaves, but this did not result in an effective biocontrol. The potential for using T. albescens for biocontrol of powdery mildews is discussed.     

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.     

Beta hydroxylation of glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-dependent β-hydroxylase

Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa, a biocontrol agent, and Ustilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis, uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δuhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δahd1 phenotype nor resulted in the production of β-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation.     

Pathogenicity of the Fungus Verticillium lecanii to Aphids and Powdery Mildew

Aspects of the host specificity and pathogenicity of the hyphomycete, Verticillium lecanii , were investigated under laboratory conditions. DiVerences were observed in the pathogenicities of three strains of V. lecanii (DAOM 198499, DAOM 216596 and Vertalec) to the potato aphid Macrosiphum euphorbiae and Sphaerotheca fuliginea , the causal agent of cucumber powdery mildew. The estimated median lethal concentration required to achieve 50% mortality (LC ), 50 median lethal time leading to 50% mortality (LT ) and aphid net reproductive rate ( R ) 50 0 indicated that Vertalec and strain 198499 were more virulent to aphids than strain 216596. The estimated median colonization time for 50% of fungal colonies (CT ) showed that strain 50 198499 was the best antagonist of cucumber powdery mildew. Further comparison suggested that the mean pathogenicity of V. lecanii strain 198499 to cucumber powdery mildew was almost equivalent to that of Sporothrix flocculosa , a biological control agent of greenhouse fungal pathogens. These observations provide convincing experimental evidence that V. lecanii is biologically active against both arthropods and fungi. The potential of using V. lecanii strain 198499 in biological control is discussed.     

Mechanisms and means of detection of biocontrol activity of Pseudozyma yeasts against plant-pathogenic fungi

Fungi belonging to Pseudozyma spp. represent a small group of yeasts that have drawn limited interest in the scientific literature. However, new research with one species of Pseudozyma, Pseudozyma flocculosa, has demonstrated the potential of this yeast as a biocontrol agent of plant-pathogenic fungi. Based on recent work, it appears that P. flocculosa, a natural inhabitant of the phyllosphere, possesses unique means of defending its ecological niche by producing unusual extracellular fatty acids that are detrimental to, among other fungi, powdery mildews, an important group of plant pathogens. Results from these studies have shown that the fatty acids naturally insert themselves into powdery mildew fungi and cause disorganization of cellular membranes and cell disintegration. Further work with insertional mutagenesis yielded mutants of P. flocculosa that represent valuable biological tools to better understand the properties of the yeast. For instance, preliminary work with mutants having lost their antagonistic properties has led to the isolation of a new metabolite with antifungal activity. Discoveries pertaining to the ecology and mode of action of P. flocculosa may lead to the study of unique metabolic or biological processes in other Pseudozyma spp. that could well release the untapped potential of these misunderstood yeasts.     

Antifungal activity of extracellular metabolites produced by Sporothrix flocculosa

The antifungal properties of extracellular compounds produced by the epiphytic fungus Sporothrix flocculosa were bioassayed against phytopathogenie fungi on the basis of inhibition of spore germination, and mycelial growth and induction of cellular leakage. Following incubation in stationary culture, S. flocculosa released antifungal metabolites into the culture medium which were extractable with méthylene chloride. When separated by thin layer chromatography, extracted metabolites yielded a compound(s) at R_f0.65 which inhibited development of Cladosporium cucumerinum and several other phytopathogenic fungi. Treatment of Botrytis cinerea and Fusarium oxysporum f.sp. radicis‐lycopersici (FORL) with the same compound(s) greatly reduced spore germination and biomass growth of both fungi. Additionally, both B. cinerea and FORL leaked electrolytes and proteins when grown in presence of the metabolites. Observations under electron microscopy revealed that FORL reacted to the presence of S. flocculosa metabolites by retraction of the plasmalemma and rapid disintegration of the cytoplasm. These reactions were similar to the ones induced by conidia of S. flocculosa when applied on powdery mildew fungi. These results provide strong evidence of the production of antifungal compounds in vivo and of their role in the antagonistic properties of S. flocculosa.     

Management of powdery mildew and gray mold of cucumber by Trichoderma harzianum T39 and Ampelomyces quisqualis AQ10

Two biocontrol preparations were tested for their ability to control Sphaerotheca fusca and Botrytis cinerea on greenhouse cucumber. Trichoderma harzianum T39 (TRICHODEX) spray reduced powdery mildew severity by up to 97% but its efficacy declined to 18–55% control as the epidemic progressed. Unlike on young leaves, on older leaves the control of powdery mildew by T. harzianum T39 was poor. Ampelomyces quisqualis (AQ10) was very effective against powdery mildew, achieving up to 98% of control. Its effectiveness declined with the progress of the epidemic but unlike the other biocontrol agent it retained significant control capability on older leaves. Two aliphatic petroleum distillate oil products improved the efficacy of both biocontrol agents. The co-application of T. harzianum T39 and A. quisqualis AQ10 was tested on cucumber plants infected with powdery mildew followed by fruit gray mold infection. It resulted in no improvement of the control of powdery mildew, and in an improvement of gray mold control, the latter probably because of the use of additive oil (ADDQ) along with the second biocontrol preparation. There was no significant interference between the biocontrol agents in the co-application treatment as compared with the application of each agent alone; the level of population of T. harzianum T39 remained similar and the parasitism of S. fusca by A. quisqualis was not nullified. The application of T. harzianum T39 to soil instead of spraying it resulted in 75–90% lower powdery mildew coverage on the leaves. It was concluded that the mode of action of T. harzianum T39 in powdery mildew control is induced resistance, not mycoparasitism or antibiotic action.     

The <Emphasis Type="Italic">Pseudozyma flocculosa</Emphasis> actin promoter allows the strong expression of a recombinant protein in the <Emphasis Type="Italic">Pseudozyma</Emphasis> species

Fungi belonging to the recently classified genus Pseudozyma possess some unique properties such as biocontrol activity, production of rare antimicrobial glycolipids and production of recombinant proteins. In this work, we report the first cloning of a promoter endogenous to the multi-faceted yeast-like Pseudozyma flocculosa, that of the actin gene. The promoter region lacked typical TATA or CAAT box but displayed three putative GC box and two CT-rich regions. As in other related basidiomycetes, only one copy of the actin gene was present in the genome of P. flocculosa. The activity of the actin promoter was compared to that of the HSP70 promoter from Ustilago maydis in two Pseudozyma species. In P. flocculosa, the actin promoter allowed the expression of a very high amount of GFP protein (27.8 mg g(-1) total protein) compared to those obtained with the HSP70 promoter in liquid culture. By contrast, the levels of GFP expression obtained in liquid culture were similar with the actin or the HSP70 promoter in Pseudozyma antarctica. A similar pattern of GFP expression was observed in solid culture. The cloning of this new promoter offers a unique genetic tool to further exploit and study the unusual properties of fungi from the Pseudozyma genus.     

亚精胺诱导黄瓜幼苗对白粉病抗性的研究

以黄瓜品种‘长春密刺’幼苗为材料,研究了亚精氨(Spd)诱导黄瓜幼苗对白粉病的抗性,并测定Spd处理和白粉菌接种对黄瓜叶片4种防御酶活性及3种防卫基因表达的影响。结果显示:(1)0.2～1.0mmol.L-1 Spd对黄瓜幼苗白粉病抗性均有不同程度的诱抗效果,并以0.8mmol.L-1 Spd处理效果最明显,诱导效率可达55.3%。(2)喷施Spd或接种白粉菌均可提高黄瓜叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶和β-1,3-葡聚糖酶的活性,且诱导并接种处理的植株叶片上述酶活性均比只诱导不接种处理的上升速度更快;同时,Spd处理和接种白粉菌可以提高植株叶片中POX、PAL、PR-1a基因的表达量。研究表明,Spd处理可以诱导防卫基因表达的增强,提高防御酶活性,显著降低病情指数,增强黄瓜幼苗对白粉病的抗性。     

Leaf and powdery mildew colonization by glycolipid-producing Pseudozyma species

In this work, we followed the development and compared the biocontrol potential of four phylogenetically related species of Pseudozyma (P. antarctica, P. flocculosa, P. fusiformata and P. rugulosa) all known to release glycolipids with antifungal activity in vitro. To this end, we developed GFP transformants and specific primers for each species and conducted in situ observations and quantification of their population sizes over time on both healthy and powdery mildew-infected leaves. Although all species shared many similar features, only P. flocculosa antagonized powdery mildew colonies and grew specifically in their presence. Population quantification by qRT-PCR following inundative applications showed a drastic decline in population for all species, including P. flocculosa on healthy leaves, but a sharp and steady increase of the latter over 72 h on infected leaves. These results suggest that production of glycolipids, and more specifically flocculosin or ustilagic acid, is not the sole factor dictating biocontrol activity among Pseudozyma spp.     

Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) as a potential mycoparasite on Sphaerotheca fuliginea (Ascomycotina: Erysiphales)

Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed.P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M.Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.     

Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15–C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.     

Oleozon: A novel control strategy against powdery mildew in cucumber

Ozonized sunflower oil (oleozon) is an effective agent for controlling powdery mildew in cucumber. In this study, the mechanisms of oleozon in the control of powdery mildew were determined. The development of Podosphaera xanthii on cucumber leaves treated with oleozon (2%) and water was investigated at different times after inoculation. The germinating conidia, hyphae and conidiophores of the pathogen were severely damaged by oleozon. No visible phytotoxic effect was observed on cucumber after the application of oleozon. This compound had highly preventive effects as well as curative effects against powdery mildew based on in vivo potted seedling assays. The control effects of oleozon were further confirmed in a greenhouse trial. These results may provide a basis for further development of a natural fungicide against cucumber powdery mildew.     

Proteomic analysis of the metabolic adaptation of the biocontrol agent <Emphasis Type="Italic">Pseudozyma flocculosa</Emphasis> leading to glycolipid production

The yeast-like epiphytic fungus Pseudozyma flocculosa is known to antagonize powdery mildew fungi through proliferation on colonies presumably preceded by the release of an antifungal glycolipid (flocculosin). In culture conditions, P. flocculosa can be induced to produce or not flocculosin through manipulation of the culture medium nutrients. In order to characterize and understand the metabolic changes in P. flocculosa linked to glycolipid production, we conducted a 2-DE proteomic analysis and compared the proteomic profile of P. flocculosa growing under conditions favoring the development of the fungus (control) or conducive to flocculosin synthesis (stress). A large number of protein spots (771) were detected in protein extracts of the control treatment compared to only 435 matched protein spots in extracts of the stress cultures, which clearly suggests an important metabolic reorganization in slow-growing cells producing flocculosin. From the latter treatment, we were able to identify 21 protein spots that were either specific to the treatment or up-regulated significantly (2-fold increase). All of them were identified based on similarity between predicted ORF of the newly sequenced genome of P. flocculosa with Ustilago maydis' available annotated sequences. These proteins were associated with the carbon and fatty acid metabolism, and also with the filamentous change of the fungus leading to flocculosin production. This first look into the proteome of P. flocculosa suggests that flocculosin synthesis is elicited in response to specific stress or limiting conditions.     

The Genera and Species Spectrum of Cucumber Powdery Mildew in Czechoslovakia

In 1979 and 1980, the fungi causing cucumber powdery mildew were investigated in Czechoslovakia. In 37 localities in the major growing 102 samples of mildew were collected. Most of them came from cucumber leaves (especially from field cultures), few came from squash leaves. Studies of the imperfect stage served to identify, in the samples, two pathogen species, viz. Erysiphe cichoracearum and Sphaerotheca fuliginea have been found. E. cichoracearum predominated under field conditions; S. fuliginea was found in only two localities. In glasshouses S. fuliginea predominated.     

Anomalies of the early stages of development of <Emphasis Type="Italic">Erysiphe graminis tritici</Emphasis> under oxidative stress

Early stage interactions between the powdery mildew pathogen and a host plant are studied. Treatment of wheat leaves with various concentrations of hydrogen peroxide and 3-amino-1,2,4-triazole resulted in the formation of morphological anomalies of germ tubes and nonviable colonies on host plant leaves. The observed effect of oxidative stress on germination anomalies of powdery mildew is similar to previously reported interactions between the pathogen and mildew resistant plants. We conclude that abnormal infectious structure formation of wheat powdery mildew may be associated with increased presence of reactive oxygen species during plant defense responses.     

Endogenous Elicitor Present in Barley Seeds

Endogenous elicitor(s) present in barley seeds induce the accumulation of antifungal substances effective against powdery mildew fungi. Antifungal substances induced by the treatment with barley seeds extract coincides with the one induced by the inoculation with compatible or incompatible races of powdery mildew fungi on thin-layer chromatography analysis. The resistance induced by the treatment with barley seeds extract correlates to the accumulation of the antifungal substances in barley leaves. Furthermore, the activity of phenylalanine ammonia-lyase was also induced by the treatment with barley seeds extract.     

Silicon-Induced changes in antifungal phenolic acids, flavonoids, and key phenylpropanoid pathway genes during the interaction between miniature roses and the biotrophic pathogen Podosphaera pannosa

Application of 3.6 mm silicon (Si+) to the rose (Rosa hybrida) cultivar Smart increased the concentration of antimicrobial phenolic acids and flavonoids in response to infection by rose powdery mildew (Podosphaera pannosa). Simultaneously, the expression of genes coding for key enzymes in the phenylpropanoid pathway (phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and chalcone synthase) was up-regulated. The increase in phenolic compounds correlated with a 46% reduction in disease severity compared with inoculated leaves without Si application (Si-). Furthermore, Si application without pathogen inoculation induced gene expression and primed the accumulation of several phenolics compared with the uninoculated Si- control. Chlorogenic acid was the phenolic acid detected in the highest concentration, with an increase of more than 80% in Si+ inoculated compared with Si- uninoculated plants. Among the quantified flavonoids, rutin and quercitrin were detected in the highest concentrations, and the rutin concentration increased more than 20-fold in Si+ inoculated compared with Si- uninoculated plants. Both rutin and chlorogenic acid had antimicrobial effects on P. pannosa, evidenced by reduced conidial germination and appressorium formation of the pathogen, both after spray application and infiltration into leaves. The application of rutin and chlorogenic acid reduced powdery mildew severity by 40% to 50%, and observation of an effect after leaf infiltration indicated that these two phenolics can be transported to the epidermal surface. In conclusion, we provide evidence that Si plays an active role in disease reduction in rose by inducing the production of antifungal phenolic metabolites as a response to powdery mildew infection.     

Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.